Total reflection fluorescent microscope

ABSTRACT

A fluorescent microscope comprises a light source, an optical illumination system which forms an optical path to irradiate a specimen with a light beam from the light source, an objective lens which condenses the light beam of the optical illumination system onto the specimen, an optical device which is disposed on the optical path of the optical illumination system and which decenters the light beam by decentering an optical axis of the optical path, and a slit which passes the light beam decentered by the optical device through a total reflection illumination region on an emission pupil surface of the objective lens.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Divisional Application of U.S. applicationSer. No. 10/848,626 filed May 18, 2004, which is based upon and claimsthe benefit of priority from prior Japanese Patent Application No.2003-143382, filed May 21, 2003, the entire contents of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a total reflection fluorescentmicroscope, which can perform fluorescent observation by totalreflection illumination.

2. Description of the Related Art

In recent years, functions of biological cells have been vigorouslyanalyzed. In the function analysis of the cells, attentions have beenpaid especially to a total reflection fluorescent microscope whichacquires a total reflection fluorescent image from a cell membrane andits vicinity as a device for observing the function of the cellmembrane.

Total reflection illumination which locally illuminates only a sample inthe vicinity of a glass surface is used in the total reflectionfluorescent microscope. In the total reflection illumination, anevanescent light is used which oozes to a sample side by about severalhundreds of nanometers in a boundary surface between glass and sample,and a background noise (scattered light and the like) is remarkably low.Therefore, fluorescent observation of even a molecular of fluorescentdyestuff is possible by the total reflection fluorescent microscope.

Additionally, in general, in the total reflection fluorescentmicroscope, a laser light beam is used as a light source. A totalreflection fluorescent microscope in which the laser light beam isintroduced into an optical illumination system of the microscope via aglass fiber is described, for example, in Jpn. Pat. Appln. KOKAIPublication No. 2002-169097.

However, the laser light source which produces the laser light beam isexpensive, and additionally a monochromatic light is produced.Therefore, for example, in order to cope with fluorescent reagentshaving various excitation wavelength characteristics, a plurality oflaser light sources have to be prepared. Therefore, the total reflectionfluorescent microscope becomes further expensive, and additionally alarge occupying space is also required for installing a plurality oflaser light sources.

To solve the problem, as described in Jpn. Pat. Appln. KOKAI PublicationNo. 2002-236258, a total reflection fluorescent microscope has beenproposed in which inexpensive white light sources such as a mercury lampand a xenon lamp are used instead of the laser light source. The totalreflection fluorescent microscope according to the Jpn. Pat. Appln.KOKAI Publication No. 2002-236258 is configured as follows. A ring slitfor transmitting a light beam in an annular form is disposed in theoptical illumination system disposed on an optical path of the lightemitted from the white laser light beam. Moreover, when an image of thering slit is projected onto an emission pupil surface of an objectivelens, an illuminative light is guided only to an orbicular totalreflection region around an emission pupil of the objective lens.Moreover, total reflection is performed in a boundary surface between aspecimen and cover glass to produce is the evanescent light, and afluorescent dyestuff is excited.

BRIEF SUMMARY OF THE INVENTION

A fluorescence microscope according to a first aspect of the presentinvention is characterized by comprising a light source; an opticalillumination system which forms an optical path to irradiate a specimenwith a light beam from the light source; an objective lens whichcondenses the light beam of the optical illumination system onto thespecimen; an optical device which is disposed on the optical path of theoptical illumination system and which decenters the light beam bydecentering an optical axis of the optical path; and a slit which passesthe light beam decentered by the optical device through a totalreflection illumination region on an emission pupil surface of theobjective lens.

A fluorescence microscope according to a second aspect of the presentinvention is characterized by comprising: a light source; an opticalillumination system which forms an optical path to irradiate a specimenwith a light beam from the light source; an objective lens whichcondenses the light beam of the optical illumination system onto thespecimen; and a slit which passes the light beam from the light sourcethrough a total reflection illumination region on an emission pupilsurface of the objective lens, in which an emission position of thelight beam emitted from the light source is movable between an opticalaxis of the optical illumination system and a position shifting from theoptical axis by a predetermined distance.

Advantages of the invention will be set forth in the description whichfollows, and in part will be obvious from the description, or may belearned by practice of the invention. The objects and advantages of theinvention may be realized and obtained by means of the instrumentalitiesand combinations particularly pointed out hereinafter.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The accompanying drawings, which are incorporated in and configure apart of the specification, illustrate embodiments of the invention, andtogether with the general description given above and the detaileddescription of the embodiments given below, serve to explain theprinciples of the invention.

FIG. 1 is a diagram showing a schematic configuration of a firstembodiment of the present invention;

FIG. 2 is a diagram showing a schematic configuration of a main part ofthe first embodiment;

FIGS. 3A to 3C are explanatory views of a slit for use in the firstembodiment;

FIG. 4 is an explanatory view of a state in which a wedge prism and slitare removed from an optical path is of an optical illumination system inthe first embodiment;

FIG. 5 is a diagram showing a schematic configuration of a main part ofa second embodiment;

FIG. 6 is a diagram showing a light flux refracted by the wedge prismfor use in the second embodiment:

FIGS. 7A to 7D are explanatory views of a slit having a crescent openingfor use in the second embodiment;

FIGS. 8A to 8E are explanatory views of a slit having a small-diameteropening for use in the second embodiment;

FIGS. 9A to 9D are explanatory views of a slit having an annular openingfor use in the second embodiment;

FIGS. 10A and 10B are diagrams showing a schematic configuration of amain part of a modification of the second embodiment;

FIG. 11 is a diagram showing a schematic configuration of a thirdembodiment of the present invention;

FIG. 12 is a diagram showing a light flux refracted by a conical prismfor use in the third embodiment;

FIG. 13 is an explanatory view of a state of a light source imageprojected on the annular opening of the third embodiment;

FIGS. 14A and 14B are diagrams showing a schematic configuration of amain part of a modification of the third embodiment;

FIG. 15 is a diagram showing a schematic configuration of a fourthembodiment of the present invention;

FIG. 16 is an explanatory view of an LED image projected on the crescentopening of the fourth embodiment;

FIG. 17 is an explanatory view of a state of an LED image projected onthe annular opening of the fourth embodiment;

FIG. 18 is a diagram showing a schematic configuration of a fifthembodiment of the present invention;

FIGS. 19A and 19B are explanatory views of a slit having a crescentopening for use in the fifth embodiment;

FIGS. 20A and 20B are explanatory views of a slit having an annularopening for use in the fifth embodiment;

FIG. 21 is an explanatory view of a slit showing a small-diameteropening for use in the fifth embodiment;

FIG. 22 is an explanatory view of a state in which an afocal converter,wedge prism, and slit are detached from an optical path of an opticalillumination system in the fifth embodiment;

FIG. 23 is a diagram showing a schematic configuration of a sixthembodiment of the present invention;

FIG. 24 is a diagram showing a schematic configuration of a seventhembodiment of the present invention; and

FIG. 25 is a diagram showing a schematic configuration of an eighthembodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention will be described hereinafter withreference to the drawings.

FIRST EMBODIMENT

FIG. 1 is a diagram showing a schematic configuration of a totalreflection fluorescent microscope to which the present invention isapplied. In this case, FIG. 1 shows an example of an inverted microscopefor performing observation by an objective lens disposed below aspecimen.

In FIG. 1, a stage 2 is disposed in an upper part of a microscope mainbody 1. A specimen 3 is laid on the stage 2. In this case, as shown inFIG. 2, a cover glass 7 is disposed under the specimen 3. An objectivelens 4 is disposed under the cover glass 7 via oil (not shown).

A revolver 5 is disposed under the specimen 3. The revolver 5 is held inthe microscope main body 1. The revolver 5 holds a plurality ofobjective lenses 4. When the revolver 5 rotates, it is possible toselectively dispose the objective lens 4 having a magnification or atype required for the observation on an observation optical axis 6. Whenthe revolver 5 vertically moves along the observation optical axis 6 byan operation of a focusing handle (not shown) to change a relativedistance between the specimen 3 and the objective lens 4 on the stage 2,the specimen 3 can be focused.

A light source 11 for illuminating the specimen 3 is used in totalreflection fluorescent illumination or usual fluorescent illumination inwhich total reflection is not performed. As the light source 11,high-luminance arc light sources such as a mercury lamp and xenon lampare used. It is to be noted that these arc light sources preferably havemicro luminescent spots, and the light source is selected having aluminescent spot in which a projected image on an emission pupil surfaceof the objective lens is smaller than an emission pupil diameter of theobjective lens.

A collector lens 12 is provided on an illuminative light axis 18 of theoptical path from the light source 11. The collector lens 12 condensesthe light beams from the light source 11 and emits a parallel lightbeam.

A wedge-shaped plane plate 41 for decentering the optical path isdisposed in the optical path of the parallel light beam from thecollector lens 12. The wedge-shaped plane plate 41 refracts the parallellight beam emitted from the collector lens 12 at a predetermined anglewith respect to the illuminative light axis 18.

A condenser 14 and a slit 15 are disposed in the optical path of a lightflux refracted by the wedge-shaped plane plate 41. The condenser 14condenses the light flux refracted by the wedge-shaped plane plate 41 ona surface of a slit 15, and a light source image 11 a of the lightsource 11 is projected.

In the slit 15, slits having openings 43 a, 43 b, 43 c having threedifferent shapes shown in FIGS. 3A to 3C are used. These openings 43 a,43 b, 43 c transmit the light beams (light source images 11 a) condensedby the condenser 14. The light beams transmitted through the openings 43a, 43 b, 43 c are to be totally reflected by a boundary surface betweenthe specimen 3 and the cover glass 7.

It is to be noted that in any of three types of slits 15, two slits aredisposed closely along an illuminative light axis 18. This is becausethe light beam that is not totally reflected, generated by framereflection inside the illuminative light axis or by a diffracted lightin the slit 15, is cut. The wedge-shaped plane plate 41 and slit 15 movealong a plane vertical to the illuminative light axis 18 by knownswitching mechanism such as a slider, and are detachably inserted withrespect to the optical path. When the wedge-shaped plane plate 41 andslit 15 are inserted into the optical path, the total reflectionfluorescent illumination can be selected. When the wedge-shaped planeplate 41 and slit 15 are removed from the optical path, the usualfluorescent illumination that does not perform the total reflection canbe selected. The slit 15 is movable along a plane crossing theilluminative light axis 18 at right angles (in a vertical direction ofan arrow shown in FIG. 1 in this case) in a state in which the slit isinserted in the optical path.

A field stop (FS) 16 and an FS projection lens 17 are disposed in theoptical path of the light transmitted through the slit 15. The fieldstop (FS) 16 is used to restrict an illumination field, and a slitdiameter can be varied. The FS projection lens 17 projects the fieldstop (FS) 16 on three surfaces of the specimen 3, and projects the imageof the slit 15 on an emission pupil surface of the objective lens 4.

A rotatable cassette 19 which holds two or more mirror units 18 a, 18 bis disposed on the optical path where the light beam is transmittedthrough the FS projection lens 17. The mirror units 18 a, 18 b aredetachably fixed to the cassette 19 by known means such as a dovetail.

The cassette 19 is rotated around a rotation axis 20. By this rotation,the mirror units 18 a, 18 b are selectively switched on the observationoptical axis 6 in accordance with wavelength characteristics of afluorescent dyestuff with which the specimen 3 is dyed. In FIG. 1, themirror unit 18 a is switched (disposed) on the observation optical axis6.

In the mirror unit 18 a, an excitation filter 211, dichroic mirror 212,and absorption filter 213 are integrally disposed as a set. Theexcitation filter 211 selectively transmits a wavelength required forexciting the specimen 3 among the light beams emitted from the FSprojection lens 17. The dichroic mirror 212 reflects an excitationwavelength from the excitation filter 211, and transmits a fluorescentwavelength emitted from the specimen 3. The dichroic mirror 212 isinclined by 45° with respect to both the illuminative light axis 18 andthe observation optical axis 6 in such a manner that an excited lightalong the illuminative light axis 18 from the excitation filter 211 isguided in a direction matching the optical axis (observation opticalaxis 6). The absorption filter 213 selectively transmits only thewavelength required for the observation in fluorescence emitted from thespecimen 3.

An optical relay system 231 is disposed in a transmission optical pathof the absorption filter 213. An image of the specimen 3 formed by theobjective lens 4 is relayed to the vicinity of an eyepiece lens 232. Theeyepiece lens 232 is used in such a manner that the image of thespecimen 3 relayed through the optical relay system 231 is visuallyobservable.

Since the mirror unit 18 b is configured in the same manner as in themirror unit 18 a, the description is omitted.

A transmission illumination section 26 includes an optical transmissionillumination system in a case where transmission illuminationobservation is performed.

A case where the total reflection fluorescent illumination is performedin the above-described configuration will be described.

When an illuminative light beam is emitted from the light source 11, thelight beam is formed into the parallel light beam by the collector lens12 and is incident upon the wedge-shaped plane plate 41.

The wedge-shaped plane plate 41 refracts the parallel light beam fromthe collector lens 12 at a predetermined angle to emit a light flux(light source image) refracted with respect to the illuminative lightaxis 18. The light flux is condensed onto the slit 15 by the condenser14.

In this case, in the slit 15, the crescent opening 43 a (or the opening43 b or 43 c) is formed linearly symmetrically with respect to a linepassing through a center of the slit 15 on a slit 15 surface as shown inFIG. 3A. A circular portion of the opening 43 a is formed substantiallyin parallel with a circumferential direction (i.e., on a substantiallyconcentric circle).

Moreover, the light source image 11 a is projected as the image of thelight source 11 in the crescent opening 43 a of the slit 15.

In the light source image 11 a projected on the slit 15, the light beamtransmitted through the opening 43 a is incident upon the excitationfilter 211 via the FS projection lens 17. The excitation filter 211selects the light beam having a wavelength required for exciting thespecimen 3. The light beam selected by its wavelength is reflectedtoward the objective lens 4 by the dichroic mirror 212, and subsequentlyprojected onto the emission pupil of the objective lens 4. It is to benoted that the image of the slit 15 projected on the emission pupilsurface of the objective lens 4, that is, a slit image will be describedlater.

All the light beams transmitted through the opening 43 a of the slit 15form totally reflected light beams within a total reflection region.

As shown in FIG. 2, the light beam transmitted through the emissionpupil surface of the objective lens 4 passes through a peripheral edgeportion of the objective lens 4, and reaches the cover glass 7 via theoil charged between the objective lens 4 and the cover glass 7. Here,the total reflection occurs in the boundary surface between the specimen3 and the cover glass 7, and the evanescent light is generated in arange of about 50 to 200 nm on a specimen side of the boundary surface.The fluorescent dyestuff with which the specimen 3 is dyed by theevanescent light is excited to emit the fluorescence.

In this state, a surveyor moves the stage 2 to search for a desiredobservation range on the specimen 3, vertically moves the revolver 5along the observation optical axis 6 by the operation of the focusinghandle (not shown), and changes the relative distance between thespecimen 3 and the objective lens 4 to focus the specimen 3.

The fluorescence emitted from the specimen 3 passes through the dichroicmirror 212, and the fluorescent wavelength required for the observationis selected by the absorption filter 213. Moreover, the image of thespecimen 3 formed by the objective lens 4 is relayed to the eyepiecelens 232 via the optical relay system 231, and visual observation ispossible.

To change the fluorescent wavelength to be observed of the fluorescentdyestuff dyed on the specimen 3, the cassette 19 is rotated around therotation axis 20 and, for example, the mirror unit 18 b may be switchedon the observation optical axis 6 instead of the mirror unit 18 a. Tochange the observation magnification of the specimen 3, the revolver 5may be rotated to position the objective lens 4 having a desiredmagnification on the observation optical axis 6.

A relation between the slit image and the emission pupil surface of theobjective lens 4 by the crescent opening 43 a of the slit 15 will bedescribed later.

In the above-mentioned description, the crescent opening 43 a is formedin the slit 15, but the small-diameter opening 43 b may also be formedin a predetermined position on the slit 15 plane, for example, as shownin FIG. 3B, the annular opening 43 c may also be formed along theperipheral edge portion of the slit 15 surface as shown in FIG. 3C, oran elliptic opening (not shown) may also be formed. A relation betweenthe slit image by the small-diameter opening 43 b, the annular opening43 c, or the elliptic opening and the emission pupil surface of theobjective lens 4 will be described later.

A case where the total reflection fluorescent observation is switched tousual fluorescent observation to perform the observation will bedescribed.

In this case, as shown in FIG. 4, the wedge-shaped plane plate 41 andslit 15 are removed from the optical path of the optical illuminationsystem, and an aperture stop (AS) 29 is inserted instead of the slit 15.Since the slit 15 is used for transmitting the illuminative light of thelight source image through a total reflection region 27 of the objectivelens 4, the slit 15 is removed from the optical path, and the aperturestop (AS) 29 is inserted as a diaphragm for adjusting brightnessinstead. The wedge-shaped plane plate 41 is used to project the lightsource image 11 a in the periphery of the emission pupil of theobjective lens 4. Especially, when the objective lens 4 has a highmagnification, and the emission pupil diameter is small, theilluminative light is reflected by the objective lens 4, the region isdarkened, and illumination unevenness sometimes occurs. Therefore, thewedge-shaped plane plate 41 is removed from the optical path.

From this state, visual sample observation is possible using a generallyknown usual fluorescent observation method.

As described above, since the wedge-shaped plane plate 41 is disposed asan optical device capable of projecting the light source image 11 a onan optical path between the light source 11 and the slit 15, the opticalaxis of the optical path is decentered and moved, for example, to theopening 43 a of the slit 15. Therefore, since the illuminative light canbe efficiently taken into the total reflection region 27 of theobjective lens 4 having an emission pupil diameter, the total reflectionfluorescent observation by sufficient brightness can be realized. In theobservation in the usual fluorescent illumination, when the wedge-shapedplane plate 41 and slit 15 are removed from the optical path, the lightsource image is projected on the optical axis. Therefore, also in thiscase, since the illuminative light can be efficiently taken in, theusual fluorescent observation by the sufficient brightness can berealized.

Moreover, the slit 15 for performing the total reflection illuminationhas the sector opening 43 b for transmitting the light beam only in apart of the total reflection region. Therefore, even when the positionor the size of the slit image changes by eccentricity of the opticalillumination system or magnification error, the slit image can beprevented from deviating from the total reflection region of theemission pupil surface of the objective lens 4. Therefore, the slitimage does not enter the fluorescent illumination region where the totalreflection is not performed, the eccentricity error can be prevented,and the total reflection fluorescent observation can be realized withgood contrast.

The opening 43 a is formed in a crescent shape to enlarge an openingarea of a portion which is not easily influenced by contrastdeterioration with respect to the eccentricity. Conversely, an openingarea of a portion easily influenced by the contrast deterioration withrespect to the eccentricity can be reduced. Therefore, since theinfluence of the contrast deterioration by the eccentricity error of theoptical system cannot be easily exerted, and additionally a middleportion of the opening 43 a has a maximum necessary opening area, theilluminative light from the light source 11 can be efficiently taken.Therefore, the total reflection fluorescent observation can be realizedwith a sufficient brightness and with good contrast and balance.

The slit 15 having the small-diameter opening 43 c is strong especiallyagainst the deterioration of the contrast by the eccentricity of theoptical illumination system. When the slit is combined with the lightsource 11 having sufficient luminance, it is easy to apply the slit evento an optical system which does not have good accuracy. Since the slit15 is easily worked, the slit is inexpensive. Furthermore, since theshape of the opening matches that of the luminescent spot of a generalhigh-luminance arc light source, the illuminative light can beefficiently taken in, and the total reflection fluorescent observationby the sufficient brightness can be realized.

The annular and elliptic openings will be explained in the secondembodiment.

Furthermore, the wedge-shaped plane plate 41 is disposed between thecollector lens 12 which projects the light source 11 as the parallellight beam and the condenser 14 which condenses the parallel light beamto form the light source image 11 a on the slit 15 plane, and eachparallel light beam in a light flux 22 is refracted by the same angle bythe wedge-shaped plane plate 41. Therefore, little aberration is causedby the wedge-shaped plane plate 41 or the condenser 14, and thesatisfactory light source image 11 a can be projected on the slit 15plane. Accordingly, the illuminative light can be efficiently taken in,and bright total reflection fluorescent observation can be performed.

SECOND EMBODIMENT

Next, a second embodiment of the present invention will be described.

FIG. 5 is a diagram showing a schematic configuration of a main part ofthe second embodiment, and the same components as those of FIG. 1 aredenoted with the same reference numerals.

In the second embodiment, a wedge prism 13 is disposed as an opticaldevice for decentering the optical axis in the optical path of theparallel light beam from the collector lens 12. The wedge prism 13refracts the parallel light beam emitted from the collector lens 12 intwo directions including upward and downward directions as shown in FIG.5, and emits the light flux 22 having a vertically linearly symmetricshape with respect to the illuminative light axis 18. FIG. 6 is adiagram concretely showing the light flux 22 refracted by the wedgeprism 13 in two vertical directions. In this case, the light flux 22refracted in two vertical directions by the wedge prism 13 is kept to beparallel.

The condenser 14 condenses the light flux 22 having two directions fromthe wedge prism 13 (parallel light beam inclined at a predeterminedangle with respect to the illuminative light axis 18) on differentplaces (two places of upper and lower places) on the slit 15 plane toproject the image of the light source 11.

The slit 15 has three types of openings 23, 24, 25 having differentshapes as described later in detail and as shown, for example, In FIGS.7A, 8A, 9A in the same manner as in the first embodiment.

Since the configuration other than the above-described configuration issimilar to that of the first embodiment, detailed description isomitted.

In this case, when the slit 15 is moved, the position of the slit imagecan be adjusted on the emission pupil surface of the objective lens 4.That is, when the slit image on the emission pupil surface is moved intoa fluorescent illumination region 28 shown in FIG. 7B, usual fluorescentobservation is possible. When the slit image is moved into the totalreflection region 27, the total reflection fluorescent observation ispossible. Furthermore, when the slit image is moved in the totalreflection region 27, an incidence angle of the illuminative light uponthe specimen from the objective lens can be finely adjusted, and anoozing depth of the evanescent light may also be controlled inaccordance with an observation position of the specimen.

The similar effect and advantage can be obtained to the first embodimenteven when the opening is formed at only either of upper or lowerportion.

The wedge prism 13 refracts the parallel light beam from the collectorlens 12 in two vertical directions as described above to emit the lightflux 22 (light source image) having a vertically linearly symmetricshape with respect to the illuminative light axis 18. The light flux 22is condensed onto two places of upper and lower places on the slit 15.

In this case, as the slit 15, it is preferable to use the slit in whichcrescent openings 23 are formed in two places of upper and lower placeshaving point symmetry with respect to a center of the slit 15 on theslit 15 plane as shown in FIG. 7A.

Moreover, the light source images 11 a are projected as the image of thelight source 11 in the crescent openings 23 of the slit 15 as shown inFIG. 7C.

FIG. 7B shows the image of the slit 15 projected on the emission pupilsurface of the objective lens 4, that is, a slit image 23 a. In FIG. 7B,an orbicular portion shown by meshes in a pupil diameter 4 a of theobjective lens 4 shows the total reflection region 27 where the light istotally reflected by the boundary surface between the specimen 3 and thecover glass 7. A shown white circular portion inside the orbicularportion shows the fluorescent illumination region 28 where the totalreflection is not performed.

Accordingly, all the light beams transmitted through the openings 23 ofthe slit 15 fall in the total reflection region 27 to form the totallyreflected light beams. Since the subsequent operation is similar to thatof the first embodiment, detailed description is omitted.

A relation between the slit image 23 a by the crescent openings 23 ofthe slit 15 and the emission pupil surface of the objective lens 4 willbe described in further detail with reference to FIG. 7B.

FIG. 7B shows a state in which the center of a slit image 23 a by thecrescent opening 23 deviates from that of a pupil diameter 4 a of theobjective lens 4. As causes for the deviation, a shift of an opticalaxis of the optical illumination system to the FS projection lens 17from the light source 11, inclination error of a reflection surface inthe dichroic mirror 212, mechanical eccentricity of the objective lens 4and the like are considered.

In consideration of a case where the slit image 23 a has the same sizeas that of the total reflection region 27, when the slit image 23 a iseccentric even slightly, a part of the slit image 23 a enters thefluorescent illumination region 28, light leak occurs, and accordingly adrop of contrast is sometimes caused. However, when the size of the slitimage 23 a is set to be smaller than that of the total reflection region27, the slit image 23 a constantly stays in the total reflection region27 even with slight movement of the slit image 23 a for theabove-described causes. Therefore, there is not fear that the light beamenters the fluorescent illumination region 28 and leaks, and theobservation with good contrast is possible. Therefore, the crescentopening 23 shown in FIG. 7A is formed in a smaller shape so as not toprotrude on a fluorescent illumination region 28 side even when the slitimage 23 a slightly moves by magnification errors of the opticalillumination system to the FS projection lens 17 from the light source11 and the magnification error of the objective lens 4.

Moreover, when the cassette 19 is rotated to insert or remove the mirrorunits 18 a, 18 b having different wavelength characteristics on theobservation optical axis 6, a positioning reproduction accuracy of arotation direction of the cassette 19 sometimes results in the error ofthe inclination of the dichroic mirror 212 or the inclination error ofthe dichroic mirror 212 for each of the mirror units 18 a, 18 b.Moreover, these errors appear as positional shifts of projection of theslit image 23 a in the objective lens emission pupil surface. In thiscase, when the slit image 23 a is configured to change its direction ina left-to-right direction with respect to these errors in FIG. 7B, theslit image 23 a by the crescent opening 23 long in the horizontaldirection constantly stays in the total reflection region 27.Accordingly, the influence of deterioration of contrast can beeliminated with respect to vibration of the slit image 23 a in thehorizontal direction. Even when the position of the opening 23 of theslit 15 slightly shifts with respect to the light source image 11 ashown in FIG. 7C, much light can be taken in from the light source 11,because the crescent openings 23 has a crescent shape long in thehorizontal direction.

It is to be noted that the slit 15 in which the crescent openings 23 areformed has been described above in detail. However, the presentinvention is not limited to this. As described in the first embodiment,for example, a slit in which small-diameter openings 24 are formed intwo positions of upper and lower positions having point symmetry withrespect to the center of the slit 15 on the slit 15 plane as shown inFIG. 8A, a slit in which annular openings 25 are formed along theperipheral edge portion of the slit 15 plane as shown in FIG. 9A, a slitin which elliptic opening (not shown) are formed and the like areconsidered.

In the slit 15 having the small-diameter openings 24, as shown in FIG.8C, the light source images 11 a by the light source 11 are projectedwith respect to the openings 24. As shown in FIG. 8B, slit images 24 aby the small-diameter openings 24 are projected on the emission pupilsurface of the objective lens 4. In the slit 15 having thesmall-diameter openings 24, the light source images 11 a sometimes shiftfrom the small-diameter openings 24 and are darkened by deviation of thepositions of the openings 24 and the light source images 11 a by theabove-described factors. However, if the light source images 11 agreatly decentered, because the opening 24 is hardly extended to thefluorescent illumination area, the contrast can be prevented beingdegraded. Since the shape of the slit 15 is simple, the slit ischaracterized in that the slit is easily worked and is inexpensive.

In the slit 15 having the annular openings 25, as shown in FIG. 9C, thelight source image 11 a by the light source 11 is projected with respectto the openings 25. As shown in FIG. 9B, a slit image 25 a by theopenings 25 is projected on the emission pupil surface of the objectivelens 4. When the slit image 25 a shifts to the right/left in the slit 15having the annular openings 25, an inner diameter of the slit image 25 aeasily overlaps with the fluorescent illumination region 28, and thecontrast easily drops. However, even when the annular openings 25 andthe light source image 11 a slightly shift, a ratio at which the lightsource image 11 a deviates from the openings 25 is small. Therefore,this is effective means for securing the brightness in a case where theeccentricity of the optical illumination system is small.

Since the shape of the opening can be matched with that of theluminescent spot of the general arc light source in the slit 15 havingthe elliptic openings shown in FIG. 8D and FIG. 8E, it is possible toefficiently take in the illuminative light.

Next, the present embodiment is similar to the first embodiment in acase where the total reflection fluorescent observation is changed tothe usual fluorescent observation to perform the observation, andtherefore the description is omitted.

As described above, according to the second embodiment, an effectsimilar to that of the first embodiment can be obtained.

It is to be noted that the size or shape of the light source 11 is notdescribed in the first and second embodiments, but the size or shape ofthe light source 11 can be set as follows.

When the slit 15 has the crescent openings 23 shown in FIG. 7A or theannular openings 25 shown in FIG. 9A, a light source for obtaining anelliptic light source image 11 b as shown in FIGS. 7D and 9D is used asthe light source 11. Moreover, the elliptic light source image 11 b isprojected on each slit 15 in a state in which the longitudinal directionis positioned transversely as shown in FIGS. 7D and 9D. Then, since thelight source image 11 b is projected on a broad range of the opening 23(25), an illumination efficiency can further be improved. To embodythis, a whole lamp house 30 in which the light source 11 is stored maybe configured so as to be rotatable around the illuminative light axis18 in accordance with the lamp shape of the light source 11. At thistime, the whole lamp house 30 may be rotatably supported, rotated by apredetermined angle in this state, and fixed via screws. Needless tosay, instead of rotating the lamp house 30, the light source 11 itselfmay be rotated in the lamp house 30.

MODIFICATION OF SECOND EMBODIMENT

Next, a modification of the second embodiment will be described.

The modification of the second embodiment is an example in which theillumination efficiency is raised without using the wedge prism, andwill be described with reference to FIGS. 10A and 10B.

As shown in FIG. 10A, the light source 11 is movable vertically in anarrow direction along the plane crossing the illuminative light axis 18at right angles. Moreover, the light source 11 can be positioned in twopositions including a position on the illuminative light axis 18 and alower position deviating slightly from the illuminative light axis 18.

To perform the total reflection fluorescent observation, the lightsource 11 is set in a position denoted with reference numeral 11′slightly deviating from the illuminative light axis 18. Then, as shownin FIG. 10B, the light beams from the light source 11′ are formed intothe parallel light beam having a predetermined angle with respect to theilluminative light axis 18 by the collector lens 12, and are projectedas a light source image 11 a′ in the opening 23 in the upper part of theslit 15. Accordingly, the illumination efficiency can be raised withoutusing the wedge prism. To return to the usual fluorescent illumination,the light source 11 may be positioned on the illuminative light axis 18.

In the present modification, the light source 11 may be moved in thevertical direction of the light source 11 with one touch. However, thelight source 11 usually has a centering function. Therefore, when thecentering function is used, the illumination efficiency can be enhancedsimply and inexpensively. It is to be noted that with the use of thewedge prism, less light is rejected by the collector lens 12 andcondenser 14. Therefore, the illumination efficiency is better that thatof the present modification, but brightness is to be enhancedinexpensively. In this case, the present modification is effectivemeans.

THIRD EMBODIMENT

Next, a third embodiment of the present invention will be described.

In the third embodiment, means for further reducing the illuminationunevenness is added to the configuration of the second embodiment.

FIG. 11 is a diagram showing a schematic configuration of the thirdembodiment.

In the third embodiment, a conical prism 31 is used instead of the wedgeprism 13 described in the second embodiment. In the conical prism 31, aconical concave portion 31 a is formed in the surface on a light source11 side, and a surface on a specimen 3 side is formed in a flat surface31 b. Moreover, the conical prism 31 is disposed in such a manner that avertex of the illuminative light axis 18 matches that of the conicalconcave portion 31 a on the optical path of the parallel light beam fromthe collector lens 12.

The conical prism 31 refracts the parallel light beam from the collectorlens 12 while keeping a parallel light flux toward the outside from theilluminative light axis 18 to emit a light flux 32. FIG. 12 is a diagramconcretely showing the light flux 32 refracted toward the outside fromthe illuminative light axis 18 by the conical prism 31. Unlike the wedgeprism 13, an inner diameter of the light flux 32 is conical.

The condenser 14 and slit 15 are disposed in the optical path of thelight flux 32 reflected by the conical prism 31. As the slit 15, a slitis used in which the annular opening 25 is formed along the peripheraledge portion as shown in FIG. 13.

In the configuration, the parallel light beam emitted from the collectorlens 12 is refracted toward the outside from the illuminative light axis18 by the conical prism 31. The refracted parallel light beam iscondensed in the annular opening 25 of the slit 15 by the condenser 14,and projected as the light source image 11 a in the opening 25 of theslit 15. In this case, an infinite number of the light source images 11a projected on the annular opening 25 of the slit 15 are projected alonga circumferential direction of the opening 25 around the illuminativelight axis 18 as shown in FIG. 13.

Moreover, the light transmitted through the slit 15 is projected as aslit image on the emission pupil surface of the objective lens 4 via theFS projection lens 17. Accordingly, the total reflection fluorescentobservation is possible in the same manner as in the second embodiment.

Thereafter, the light flux 32 refracted toward the outside from theilluminative light axis 18 is generated by the conical prism 31, andaccordingly the light source image 11 a can be projected along theannular opening 25 of the slit 15. Accordingly, in addition to theeffect of the second embodiment, since the annular opening 25 can beuniformly illuminated, the illumination unevenness can be largelyreduced.

MODIFICATION OF THIRD EMBODIMENT

Next, a modification of the third embodiment will be described

The modification of the third embodiment is an example including anothermeans for reducing the illumination unevenness, and will be describedwith reference to FIGS. 14A and 14B.

In this case, the wedge prism 13 is disposed in the optical path betweenthe collector lens 12 and the condenser 14 in the same manner as in thesecond embodiment. Moreover, a slit in which the annular opening 25 isformed along the peripheral edge portion as shown in FIG. 14B is used asthe slit 15. Further-more, in this state, the wedge prism 13 is rotatedat a high speed in a direction of an arrow 33 using the illuminativelight axis 18 which is a rotational center. In this case, the prism isrotated once at about 30 msec in the visual observation, or rotated at arotation number higher than a scanning speed of photo-detection, whenphoto-detection means such as CCD. Accordingly, as shown in FIG. 14B,the light source image 11 a rotates along the annular opening 25 aroundthe illuminative light axis 18. Therefore, when a time average of therotation is taken, an effect similar to that with the use of the conicalprism 31 described in the third embodiment is obtained. In this case,the rotation means of the wedge prism 13 can be realized using a knownmotor and bearing.

Moreover, the light transmitted through the slit 15 is projected on theslit image on the emission pupil surface of the objective lens 4 via theFS projection lens 17. Accordingly, the total reflection fluorescentobservation is possible in the same manner as in the second embodiment.

Therefore, when the wedge prism 13 is configured so as to be rotatablearound the illuminative light axis 18 of the optical illumination systemat a high speed, the light source image 11 a can be projected along theannular opening 25, and therefore the total reflection fluorescentobservation is realized with the illumination without any directionalityor unevenness. The cost can also be reduced without using the expensiveconical prism 31.

FOURTH EMBODIMENT

A fourth embodiment of the present invention will be described.

FIG. 15 is a diagram showing a schematic configuration of the fourthembodiment, and the same components as those of FIG. 1 are denoted withthe same reference numerals.

In the fourth embodiment, six LEDs 34 having micro luminescent spots aredisposed instead of the light source 11. In this case, six LEDs 34 aredisposed in the position of the point symmetry with respect to theilluminative light axis 18 on the plane crossing the illuminative lightaxis 18 at right angles. In the fourth embodiment, the wedge prism 13 isnot required. As the slit 15, a slit is used in which the crescentopenings 23 are formed in two positions of upper and lower positions ofthe point symmetry with respect to the center of the slit 15 on the slit15 plane as shown in FIG. 16.

In the above-described configuration, the illuminative lights emittedfrom six LEDs 34 are formed into parallel light beams having apredetermined angle with respect to the illuminative light axis 18 bythe collector lens 12, and are projected as LED images 35 in theupper/lower crescent openings 23 of the slit 15 by the condenser 14 asshown in FIG. 16.

Moreover, the light transmitted through the slit 15 is projected as theslit image on the emission pupil surface of the objective lens 4 via theFS projection lens 17. Accordingly, the total reflection fluorescentobservation is possible in the same manner as in the second embodiment.

Therefore, since the respective LED images 35 of six LEDs 34 can beprojected on accordance with the upper/lower crescent openings 23 of theslit 15, the illumination efficiency can be further enhanced.

It is to be noted that when the slit having the annular openings 25 asshown in FIG. 17 is used as the slit 15, a large number of LEDs 34 arearranged in an annular form around the illuminative light axis 18.Moreover, the lights from the LEDs 34 arranged in the annular form areprojected as the LED images 35 in the annular openings 25 of the slit 15via the collector lens 12 and condenser 14.

Since the LED images 35 from the annularly arranged LEDs 34 can beuniformly projected along the annular openings 25 of the slit 15 in thismanner, the illumination unevenness can be reduced.

It is to be noted that when the number of LEDs 34 is further increasedand a large number of LEDs are arranged around the illuminative lightaxis 18, the LEDs 34 can only be selectively lit in accordance with theshapes of the openings of the slit 15 to project the LED images 35 inaccordance with various openings. All the LEDs 34 may be lit in theusual fluorescent illumination observation.

According to the fourth embodiment, by the use of the light sourcehaving a plurality of micro luminescent spots arranged to fill theopenings 23 (25) of the slit 15, the light source image is projectedonly in a range passing through the openings 23 (25), and theilluminative light is not introduced except the total reflection region.Therefore, the illuminative light can be efficiently taken in, and thetotal reflection fluorescent observation is possible with the sufficientbrightness and good contrast. Since the expensive wedge prism or conicalprism is not used, the microscope is inexpensive. Especially, when aslit having the annular opening 25 is used as the slit 15, the totalreflection fluorescent illumination having remarkably little unevennessmay also be obtained.

FIFTH EMBODIMENT

A fifth embodiment of the present invention will be described.

FIG. 18 is a diagram showing a schematic configuration of the fifthembodiment, and the same components as those of FIG. 1 are denoted withthe same reference numerals.

In the fifth embodiment, an afocal converter 36 is disposed asmagnification varying means for raising light source magnification inthe optical path between the collector lens 12 and the wedge prism 13.The afocal converter 36 comprises a convex lens 36 a and concave lens 36b. By the afocal converter 36, the parallel light beam from thecollector lens 12 is condensed onto the convex lens 36 a, and divertedby the concave lens 36 b. Accordingly, the parallel light beam whoselight source magnification has been raised can be emitted. The afocalconverter 36 is detachably inserted together with the wedge prism 13 andslit 15 with respect to the optical path.

In the slit 15, a slit in which the crescent openings 23 are formed inthe two positions of upper and lower positions of the point symmetrywith respect to the center of the slit 15 on the slit 15 plane as shownin FIGS. 19A and 19B, or a slit in which the annular openings 25 areformed along the peripheral edge portion of the slit 15 as shown inFIGS. 20 and 20B are used.

When the total reflection fluorescent illumination is performed, theilluminative light emitted from the light source 11 is projected as alight source image 37 on the slit 15 plane via the collector lens 12,the convex lens 36 a and concave lens 36 b configuring the afocalconverter 36, the wedge prism 13, and the condenser 14. In this case, inthe light source image 37, since the light source magnification israised by the afocal converter 36, the light source image 37 projectedon the slit 15 plane spreads sufficiently in a broad range on therespective openings 23, 25 as shown in FIG. 19B or 20B. FIG. 19A or 20Ashows a case where the afocal converter 36 is not disposed, and thelight source image 37 projected on the slit 15 plane overlaps with apart of the openings 23, 25.

Therefore, to perform the total reflection fluorescent illumination,when the convex lens 36 a and concave lens 36 b configuring the afocalconverter 36 are inserted in the optical path to raise the magnificationof the light source image 37, more openings 23 (25) of the slit 15 canbe filled with the light source images 37, and therefore theillumination efficiency can be further enhanced.

The light transmitted through the slit 15 is projected as the slit imageon the emission pupil surface of the objective lens 4 via the FSprojection lens 17, and the total reflection fluorescent observation ispossible in the same manner as in the second embodiment.

When the small-diameter openings 24 are formed in two positions of upperand lower positions of the point symmetry with respect to the center ofthe slit 15 in the slit 15 as shown in FIG. 21, even the light sourceimages 37 projected onto the slit 15 plane sufficiently fill theopenings 24 of the slit 15 in a state free of the afocal converter 36.Therefore, even when the light source magnification is raisedparticularly using the afocal converter 36, an effect of enhancement ofthe illumination efficiency is little.

Next, a case where usual fluorescent observation is performed will bedescribed.

As shown in FIG. 22, the afocal converter 36, wedge prism 13, and slit15 are removed from the optical path of the optical illumination system,and the aperture stop (AS) 29 is inserted instead of the slit 15. Whenthe wedge prism 13 or the slit 15 enters the optical path, theillumination efficiency drops or the illumination unevenness increasesin the same manner as in the second embodiment. Therefore, to performthe usual fluorescent observation, the prism or the slit is removed fromthe optical path, and the aperture stop (AS) 29 for adjusting thebrightness is inserted instead of the slit 15. This can be realized bythe use of an inserting/detaching mechanism such as a known slider. Theconvex lens 36 a and concave lens 36 b configuring the afocal converter36 are effective for enhancing the illumination efficiency. However, onthe contrary, the illumination field is narrowed, and an observablerange is narrowed. Therefore, the afocal converter 36 is not required atthe time of the usual fluorescent observation with the sufficientbrightness, and is also removed from the optical path.

It is to be noted that the afocal converter 36 is used to changing thelight source magnification. Therefore, even when the converter isremoved from the optical path, a projection plane of the light source 11is unchanged. Therefore, even when the aperture stop (AS) 29 is insertedin the optical path instead of the slit 15, the light source image isprojected on the aperture stop (AS) 29 plane. Therefore, there is nofear that the illumination unevenness occurs also at the usualfluorescent observation time, the illumination efficiency does not drop,the illumination is bright, and therefore optimum microscopic inspectioncan be performed.

In this state, specimen can be visually observed by the generally knownusual fluorescent observation method.

It is to be noted that another variable magnification lens is alsousable as means for raising the light source magnification in additionto the afocal converter 36.

In the fifth embodiment, the afocal converter 36 is disposed asmagnification varying means for raising a projection magnification ofthe light source 11 between the slit 15 and the light source 11.Accordingly, in the total reflection illumination, the magnification ofthe light source image is raised, and even a portion incapable offilling the openings 23 of the slit 15 is filled with the light sourceimage 37. Accordingly, the total reflection fluorescent observation ispossible by brighter illumination. Even when the afocal converter 36 isinserted or removed with respect to the optical path, the projectionposition of the light source image 37 in the optical-axis direction doesnot change. Therefore, the illumination efficiency at a total reflectionfluorescent observation time does not drop. Moreover, the illuminationunevenness does not easily occur at a usual fluorescent observationtime, and optimum microscopic inspection can be performed in eachobservation.

SIXTH EMBODIMENT

A sixth embodiment of the present invention will be described.

FIG. 23 is a diagram showing a schematic configuration of a main part ofa sixth embodiment, and the same components as those of FIG. 1 aredenoted with the same reference numerals.

In this case, the collector lens 12, condenser 14, and slit 15 aredisposed on the illuminative light axis 18 of the light from the lightsource 11. A convex lens 44 having a large diameter is disposed as alens having a weak refractive power between the collector lens 12 andthe condenser 14.

The convex lens 44 is disposed while a central axis 44 a is largelyshifted from the illuminative light axis 18, and the parallel light beamfrom the collector lens 12 is refracted by a predetermined angle withrespect to the illuminative light axis 18. The condenser 14 condenses alight flux 45 refracted by the convex lens 44 is condensed on the slit15 plane, and the light source image 11 a is projected. Also in thiscase, a slit is used in which a crescent opening 43 is formed on theslit plane is used in the same manner as in FIG. 3A. The light sourceimage 11 a is projected on the crescent opening 43 a via the condenser14.

Also in the sixth embodiment, the convex lens 44 and slit 15 can bedetachably inserted with respect to the optical path of the illuminativelight by known switching mechanisms such as a slider. The slit 15inserted in the optical path is movable further along the plane crossingthe illuminative light axis 18 at right angles in a known arrowdirection.

The other configuration is similar to that of FIG. 1.

Therefore, the effect similar to that of the first embodiment can beexpected.

SEVENTH EMBODIMENT

A seventh embodiment of the present invention will be described.

FIG. 24 is a diagram showing a schematic configuration of a main part ofthe seventh embodiment, and the same components as those of FIG. 1 aredenoted with the same reference numerals.

In FIG. 24, the collector lens 12, condenser 14, and slit 15 aredisposed on the illuminative light axis 18 of the light from the lightsource 11. A parallel plane plate 46 is disposed between the condenser14 and the slit 15.

The parallel plane plate 46 is inclined at a predetermined angle withrespect to the illuminative light axis 18 and disposed to move the lightbeam from the condenser 14 in parallel with the illuminative light axis18, and condenses the light on the slit 15 plane to project the lightsource image 11 a. Also in this case, a slit in which the crescentopening 43 is formed on the slit plane is used as the slit 15 in thesame manner as in FIG. 22. The light source image 11 a is projected onthe crescent opening 43 via the condenser 14.

Also in this case, the parallel plane plate 46 and slit 15 aredetachably inserted with respect to the optical path of the illuminativelight by known switching mechanisms such as the slider. Moreover, theinclination angle of the parallel plane plate 46 inserted in the opticalpath is adjustable in the arrow direction. Therefore, even when thetotal reflection region differs with the type of the objective lens, theinclination angle of the parallel plane plate 46 can be adjusted toadjust the light source image 11 a in an optimum position in the totalreflection region. Furthermore, even the slit 15 inserted in the opticalpath can move along the plane crossing the illuminative light axis 18 atright angles in the arrow direction.

The other configuration is similar to that of FIG. 1.

Therefore, the effect similar to that of the first embodiment can beexpected.

EIGHTH EMBODIMENT

An eighth embodiment of the present invention will be described.

FIG. 25 is a diagram showing a schematic configuration of a main part ofthe eighth embodiment, and the same components as those of FIG. 1 aredenoted with the same reference numerals.

In FIG. 25, the collector lens 12, condenser 14, and slit 15 aredisposed on the illuminative light axis 18 of the light from the lightsource 11. Mirrors 47, 48 are disposed between the condenser 14 and theslit 15.

The light beam from the condenser 14 is reflected by the mirror 47, andthe reflected light is reflected by the mirror 48. Accordingly, theoptical path of the optical illumination system is moved in parallel,that is, the light beam from the condenser 14 is moved in parallel withthe illuminative light axis 18 and condensed onto the slit 15 plane toproject the light source image 11 a. Even in the eighth embodiment, theslit in which the crescent opening 43 is formed on the slit plane asdescribed with reference to FIG. 22 is used as the slit 15. The lightsource image 11 a is projected on the crescent opening 43 via thecondenser 14.

Even in this case, the mirrors 47, 48 and the slit 15 can be detachablyinserted with respect to the optical path of the illuminative light bythe known switching mechanisms such as the slider. In the mirrors 47,48, the mirror 48 is movable in the arrow direction, and the distancefrom the mirror 47 can be adjusted. Accordingly, even when the totalreflection region differs with the type of the objective lens, thedistance between the mirrors 47, 48 can be adjusted so as to adjust thelight source image 11 a in an optimum position in the total reflectionregion. Furthermore, even the slit 15 inserted in the optical path canmove along the plane crossing the illuminative light axis 18 at rightangles in the arrow direction.

The other configuration is similar to that of FIG. 1.

Therefore, the effect similar to that of the first embodiment can beexpected.

The present invention is not limited to the above-described embodiments,and can be variously modified in a range in which the scope is notchanged. For example, in the embodiments, the optical device fordecentering the optical axis has been described. Alternatively, aplurality of optical devices having different eccentricities of theoptical axis are prepared, and may also be selectively used inaccordance with the type of the objective lens (the total reflectionregion differs). The plane of the slit 15 on the light source 11 sidemay also be formed in a reflection plane or an irregular reflectionplane. In this case, degradation by heat or the like in a portion whichinterrupts the light beam of the slit 15, that is, a portion irradiatedwith the light beam can be reduced. Furthermore, in the embodiments, theinverted microscope has been described by which the observation isperformed by the objective lens disposed under the specimen, but atransmission illumination type may also be used in which the totalreflection fluorescent illumination is performed using a condenser lens,or an erected microscope may also be used.

Furthermore, the embodiments include various stages of inventions, andvarious inventions can be extracted by an appropriate combination of aplurality of configuring elements. For example, even when severalconfiguring elements are removed from all the configuring elementsdescribed in the embodiments, the described problems to be solved by thepresent invention can be resolved, and the described effects of thepresent invention are obtained. In this case, the configuration fromwhich the configuring elements are removed can be extracted as theinvention.

It is to be noted that the above-described embodiments also include thefollowing inventions.

A fluorescence microscope according to a first aspect of the presentinvention is characterized by comprising a light source; an opticalillumination system which forms an optical path to irradiate a specimenwith a light beam from the light source; an objective lens whichcondenses the light beam of the optical illumination system onto thespecimen; an optical device which is disposed on the optical path of theoptical illumination system and which decenters the light beam bydecentering an optical axis of the optical path; and a slit which passesthe light beam decentered by the optical device through a totalreflection illumination region on an emission pupil surface of theobjective lens. In the first aspect, the following manners arepreferable.

(1) The total reflection illumination is illumination using anevanescent light oozing by a predetermined amount on a specimen side ina boundary surface between glass on which the specimen is laid and thespecimen.

(2) The optical device and the slit are movable along a plane verticalto the optical axis of the optical path of the optical illuminationsystem.

(3) The opening has at least one of a crescent shape, a circular shape,a half-ring shape, and an elliptic shape.

(4) The optical device is a prism.

(5) In (4), the prism is either a wedge prism or a conical prism.

(6) In (4), the prism is a wedge-shaped plane plate.

(7) The optical device is a lens having a small refractive index.

(8) The optical device is a parallel plane plate disposed with apredetermined angle with respect to the optical axis.

(9) The optical device comprises a pair of mirrors which move theoptical path of the optical illumination system.

(10) The optical device includes a plurality of optical devices, and theplurality of optical devices are selectively inserted in the opticalpath of the optical illumination system.

(11) In (5) or (6), the wedge prism is rotatable around the optical axisof the optical illumination system.

(12) The light source has a luminescent spot in which a projected imageon an emission pupil surface of the objective lens is smaller than anemission pupil diameter of the objective lens.

(13) In (12), the light source is either a micro arc type light sourceor a light source comprising a plurality of micro luminescent spots.

(14) In (13), the light source comprising the plurality of microluminescent spots includes a plurality of light emitting diodes.

(15) In (14), the plurality of light emitting diodes are arranged on acircumference having a predetermined diameter.

(16) An optical magnification varying system which is disposed in theoptical path of the optical device on the light source side to raise aprojection magnification of the light source is further provided.

(17) In (16), the optical magnification varying system includes anafocal converter.

(18) The slit has a reflection surface or an irregular reflectionsurface formed on a plane on a light source side.

A fluorescence microscope according to a second aspect of the presentinvention is characterized by comprising: a light source; an opticalillumination system which forms an optical path to irradiate a specimenwith a light beam from the light source; an objective lens whichcondenses the light beam of the optical illumination system onto thespecimen; and a slit which passes the light beam from the light sourcethrough a total reflection illumination region on an emission pupilsurface of the objective lens, in which an emission position of thelight beam emitted from the light source is movable between an opticalaxis of the optical illumination system and a position shifting from theoptical axis by a predetermined distance.

According to the embodiments of the present invention, the opticaldevice for decentering the optical axis of the optical path is disposedbetween the light source and the slit. Accordingly, the illuminativelight can be efficiently taken in the total reflection region having anemission pupil diameter of the objective lens by the eccentricity of theoptical axis of the optical path.

Moreover, the optical device and slit are detachably inserted in theoptical path. Therefore, when the states are only selected, theilluminative light can be efficiently taken into the total reflectionregion of the emission pupil surface of the objective lens or thefluorescent illumination region where the total reflection is notperformed.

Furthermore, the slit for performing the total reflection illuminationhas the crescent opening which passes the light beam only through a partof the total reflection region. Accordingly, even when the position orthe size of the slit image changes by the eccentricity or themagnification error of the optical illumination system, the slit imagecan be prevented from deviating from the total reflection region of theemission pupil surface of the objective lens.

Moreover, since the wedge prism or the conical prism is used as theoptical device, the respective parallel light beams in the light fluxcan be refracted by an equal angle, little aberration is generated bythe prism or the condenser, and a satisfactory light source image can beprojected on the slit plane.

Furthermore, since a light source having a plurality of microluminescent spots is used as the light source, the light source image isprojected only in a range passing through the slit. Since theilluminative light is not introduced into a region other than the totalreflection region, the illuminative light can be efficiently taken.

Moreover, since the wedge prism is rotatable around the optical axis ofthe optical illumination system, the light source image can be projectedon the annular shape, and the illumination is obtained without anydirectionality or unevenness.

Furthermore, since an optical magnification varying system for changinga projection magnification is disposed in the optical path of theoptical device on the light source side, in the total reflectionillumination, the magnification of the light source image is raised, andeven the portion of the slit that cannot be filled is filled with thelight source image, and the total reflection fluorescent observation bybrighter illumination is possible.

As described above, according to the embodiments of the presentinvention, there can be provided a total reflection fluorescentmicroscope in which use efficiency of the illuminative light is raised,and the total reflection fluorescent observation is possible with thesufficient brightness and good contrast.

Additional advantages and modifications will readily occur to thoseskilled in the art. Therefore, the invention in its broader aspects isnot limited to the specific details and representative embodiments shownand described herein. Accordingly, various modifications may be madewithout departing from the spirit or scope of the general inventionconcept as defined by the appended claims and their equivalents.

1. A fluorescent microscope comprising: a light source; an opticalillumination system which forms an optical path to irradiate a specimenwith a light beam from the light source; an objective lens whichcondenses the light beam from the light source, which has traveledthrough the optical illumination system, onto the specimen; and a slitwhich is located in the optical illumination system to pass the lightbeam from the light source through a total reflection illuminationregion on an emission pupil surface of the objective lens, wherein anemission position of the light beam emitted from the light source ismovable between an optical axis of the optical illumination system and aposition shifted from the optical axis by a predetermined distance. 2.The fluorescent microscope according to claim 1, wherein the opticalillumination system comprises: a collector lens which parallelizes thelight beam from the light source; a condenser which condenses the lightbeam from the light source that has passed through the collector lens;and a projection lens which projects an image formed by the light beamcondensed by the condenser onto an emission pupil surface of theobjective lens.
 3. The fluorescent microscope according to claim 1,wherein the slit is removed from the optical illumination system, andthe emission position of the light beam emitted from the light source ismoved onto the optical axis of the optical illumination system, toperform fluorescent illumination.
 4. A fluorescent microscopecomprising: an objective lens which is used in observation of aspecimen; a movable light source which irradiates the specimen with alight beam; a collector lens which parallelizes the light beam from thelight source; a condenser which condenses the light beam from the lightsource that has passed through the collector lens; and a slit which islocated at a position onto which the light beam is condensed by thecondenser; a projection lens which projects an image formed by the lightbeam from the light source through the slit onto an emission pupilsurface of the objective lens, wherein when the light source is moved toa position displaced at least from an optical axis of the collectorlens, the image formed by the light beam from the light source isprojected onto the slit, and the light beam from the light source iscaused to pass through a total reflection illumination region of theemission pupil surface of the objective lens.
 5. The fluorescentmicroscope according to claim 4, wherein the light source is moved ontothe optical axis of the collector lens and the slit is removed from apath of the light beam from the light source to perform fluorescentillumination.